Protein inhibitors, which may function in different ways to “inhibit” a protein (e.g. by inhibiting its synthesis or its activity), are widely used as drugs to combat diseases. The diseases may be caused by overexpression or overactivity of the protein in question, as in the case in a number of cancers where the overexpression of certain oncogenes is involved with the development of malignancies e.g. HER2/neu in breast cancer, the ras oncogene and the myc oncogene. The overexpression may cause overactivity, or overactivity may result from mutation. Oncogene proteins are frequently components of a pathway, such as a signalling pathway, that is important in the regulation of cell growth. Once a protein is shown to be required for the cause or progress of a disease state, it is desirable to “target” the protein and attempt to find drugs that act to prevent the disease-related function of the protein.
The disease may also be an infection, such as a fungal or bacterial infection. Such diseases may be combatted with antibiotics, which again act to inhibit the function of a “target” protein as infection introduces new proteins into the infected cells. These proteins that are not found in the uninfected host cell may be target proteins.
Infectious diseases are a major cause of mortality worldwide and as such, new agents that are useful to act against infection are required. However, the overuse of antibiotics in particular has led to the phenomenon of antibiotic resistance where antibiotics become ineffective against microorganisms and it is therefore of crucial importance to develop new and improved antibiotics. It is particularly important to identify antibiotics that are chemically distinct from those that are currently used. Microorganisms that exhibit antibiotic resistance to a certain antibiotic are more likely to show resistance to one that is chemically related, than one which is structurally and functionally distinct. Therefore, new antimicrobial compounds that are identified based on their function, rather than their structure, may be of particular use. The development of antibiotics that act against targets for which no known antibiotics exist is also of particular interest.
Current antibiotic targets include enzymes involved in protein synthesis and membrane transporters or cell wall components. These targets are currently identified in a number of ways. The huge increases in nucleic acid sequence data that is available has led to an increase in the ability to attribute a function to a protein based on the comparison of sequence data. The sheer amount of data available however makes this difficult and about 25-40% of the genes in a bacterial genome will not have matches with counterpart, known genes (Smith D. R. (1996) Trends Biotechnology 8: 290-3). In addition, the fact that two genes share sequence homology does not always mean that they will be structurally similar.
Drug targets, whether for antibiotics or against other diseases, should ideally have the following properties: they must be necessary for the pathogen or disease to survive, grow or act; in relation to antibiotic targets, or anti-pathogenic targets it is also useful that the target protein is absent or distinct in humans, or the mammal which is to be treated; and the degree of conservation of structure of the drug target between species that are being combatted is preferably high. To date, no drugs that target the DNA replication machinery have been identified.
Once a target protein has been identified, it is necessary to identify compounds that may act to inhibit or impede its function, whether in a disease state or in the pathogen. The process of screening for inhibitory compounds, which previously has been labourious and time consuming has been improved by technologies that allow high throughput screening where many hundreds or even thousands of compounds may be tested simultaneously or in parallel.
In general, such screens to identify antibiotics or other protein inhibitors are “negative” screens. In these screens, a protein inhibitor is identified following the application of a test substance to a cell population, when the cell population exhibits a reduction in viability. This follows from the fact that one of the properties of the target outlined above is that it is essential to the continuing growth and proliferation of the pathogen in question. Interfering with this essential function affects cell viability. Thus, most screening techniques rely on a negative result ensuing from the abrogation of function of the target, such as the death or reduction in growth of cells containing and requiring it. This approach suffers from several disadvantages. Several different assays must be performed, sequentially or in parallel, to ensure that the observed effect on cell viability is specific to the target protein. The fact that the effect, e.g. cell death alone is seen is not sufficient to confirm that the effect is caused by an effect on the target protein. The test substance may have affected a different target protein or the effect may be a general effect that is entirely unrelated to the specific target protein. Thus, such negative screens are time consuming and it is not possible to obtain a result without performing multiple assays.
It would be advantageous to have an assay or screen for protein inhibitors that depends on a positive result or outcome, such as increased cell growth or survival of a mutant cell that is unable to grow otherwise. Such positive screens are advantageous as they avoid many of the problems associated with negative screens, such as high background levels of cell death or lack of growth that may not be attributable to the specific action of a potential inhibitor, but may be due to other reasons. No such positive screens are presently known in the art. The present invention now provides such a test.
Lethal overactivity mutants are mutants that contain mutation(s) such that the activity of a particular gene product produced or expressed is greater, compared to the wild-type organism or the “non-mutated” gene product (i.e. the gene product prior to the introduction of the lethal over-activity mutation—it is not precluded that the gene product may carry other mutations not related to the lethal overactivity). This may be due to differences in the activity of the gene product itself, in its levels of expression or production, or in the regulation of its activity (e.g. due to an increase or decrease in the expression and/or activity of a regulatory molecule). Thus a lethal overactivity mutant may be viewed as one in which the “functionality” of the gene product concerned is increased. The increase in activity (or “functionality”) of this gene product is detrimental to the survival and/or growth of the mutant cell. The gene product may thus be any product which is lethal, or which has a significant negative effect on the growth and/or survival of the organism, when its activity exceeds certain levels (e.g. “normal” levels e.g. prior to the mutation, or native or wild-type levels) e.g. when it is “over-active”. Such over-activity may, as mentioned above, be achieved in various ways, including by an increase in amount or content, or levels of the gene product e.g. due to increased expression or reduced breakdown, or by increased or prolonged activity. Such mutants are therefore useful in positive screens. However such mutations are rare in nature and can be difficult to generate.
DnaA is an eubacterial protein that initiates chromosomal replication in bacteria (Kornberg and Baker 1992, DNA replication, W.H. Freeman). It is at this stage that the cycle of chromosomal replication is regulated. DNA replication is also dependent on the presence of a unique chromosomal sequence, OriC the replication origin. Both OriC and DnaA are required for successful initiation of replication, and these two components form a nucleoprotein complex. About 20-40 monomers of DnaA protein are present in the OriC-DnaA complex, ATP-bound DnaA causes the DNA duplex to start to unwind, thus allowing the DnaB helicase to extend the unwinding, prior to synthesis of the complementary strands by DNA polymerase III holoenzyme (Skarstad and Boye, Biochim. Biophys. Acta, 1994, 1217, 111-130, reviewed in Katayama et al., Molecular Microbiology, 2001, 41(1), 9-17).
Initiation of DNA replication in prokaryotes and eukaryotes is highly regulated by a number of mechanisms, due to its importance in the cell cycle. Excessive initiation events eventually lead to cell death.
One example of a mutation that causes hyperactive initiation is DnaAcos, which is a mutation identified in E. coli (Kellenberger-Gujer et al., Molec. Gen. Genet. 162, 9-16, 1978; and Katayama et al. (1994), Journal of Biological Chemistry, 269(17), 12698-12703). This mutant was isolated as a temperature resistant suppressor from a temperature sensitive DnaAts46 mutant. DnaAts46 is a well-characterised E. coli mutant which expresses a DnaA protein which is inactive at elevated temperatures (42° C.), resulting in a mutant strain which is unable to initiate replication at elevated temperatures (Kohiyama, Cold Spr. Harb. Symp. Quant. Biol. 33, 312-324 (1968); Hirota et al., J. Molec. Biol. 53, 369-387 (1970).
The DnaAcos mutant is known to have the following properties. Firstly, its growth is cold sensitive; it grows normally at 42° C., however replication of chromosomal DNA over-initiates immediately once the cells are shifted to grow at the restrictive temperature of 30° C. DnaAcos has been identified as a suppressor mutant of dnaAts46 i.e. it suppresses the dnaAts46 phenotype and represents an intragenic suppressor mutant. The suppressor mutations (Q156L and Y271H) result from base substitutions in the dnaA gene (Hansen et al., 1992, Mol. Gen. Gent., 234, 15-21, Skarstad and Boye, 1994, Biochim Biophys Acta, 1217, 111-130, Kellenbergen-Gujer et al., 1978, Mol. Gen. Genet., 162, 9-16).
The cold sensitivity of dnaAcos is dominant over the wild-type dnaA allele, and the over-initiation seen at the restrictive temperature is independent of de novo protein synthesis. Interestingly, there is no increase in the amount of DnaA protein in this mutant and the mutant phenotype is thought to depend on increased and/or prolonged DnaA activity. Since initiation with the mutant occurs repeatedly it has been suggested that somehow the initiation competence of the mutant DnaAcos protein is sustained, e.g. through a conformational change.
The mechanism of over-initiation in the mutant is not fully understood, although the DnaAcos protein has been purified and characterised in vitro (Katayama et al., 1995, Mol. Microbiol., 18, 813-820). It sustains affinity to a DnaA-binding sequence and functions in the loading of DnaB helicase onto single-stranded DNA. The purified wild type DnaA protein binds ATP and ADP. However, the DnaAcos protein is unable to bind nucleotide. Wild type ATP-DnaA is active in initiation of replication while wild-type ADP-DnaA is inactive (Sekimizu et al., 1987, Cell, 50, 259-265). The hydrolysis of wild-type ATP-DnaA to ADP-DnaA inactivates DnaA to regulate its function and this occurs as soon as replication forks are underway to prevent reinitiation of an already initiated origin (Boye et al., 2000, EMBO Rep. 1, 479-483). The DnaAcos protein seems to be an “unregulated” form of DnaA protein that is always “turned on” and therefore causes excess DNA replication at lower temperatures (30° C.). At higher temperatures (42° C.) the protein is apparently partially inactive, explaining that the over-initiation is reduced compared to the restrictive temperature, and the cells therefore survive.
Other DnaA mutants have been, or may be, developed which are similar to, or have the properties of DnaAcos (e.g. which exhibit temperature sensitive replication) e.g. over-inhibition of replication at lower temperature (e.g. 30° C.). One such mutant is DnaA219 used in the Examples herein.
DnaA and homologues of this protein in other prokaryotes or in eukaryotes or Archaea, represent an example of a target for a protein inhibitor, as it is an essential protein for E. coli and is also highly conserved between different bacterial species. Other DNA replication initiator proteins (whether eukaryotic, prokaryotic or archael) may also represent protein inhibitor targets.
In a comparison of 104 sequences from 96 species, DnaA was shown to have a highly conserved primary sequence, and the overall arrangement of 15 α helices and 9 β strands seen is over 93% of the sequences (Weigel and Messer 2002, on the World-Wide Web at molgen.mpg.de/˜messer). Furthermore, the Cdc6 and Orc initiator proteins of yeast, e.g. Saccharomyces cerevisiae shows striking structural similarity to DnaA and thus represent a eukaryotic target protein (Erzberger et al. (2002) EMBO J 21: 4763-73, Liu et al., (2000) Mol. Cell. 6: 637-48).
Overactivity mutants of target proteins, e.g. DnaA mutants that over-initiate DNA replication, can be used to assay for potential new protein inhibitors; any protein inhibitor that interferes with the function of the target protein (e.g. DnaA) will reduce the amount of over-initiation and thereby increase the growth of the population of mutant cells compared to their growth in the absence of such a protein inhibitor. However such an assay would only allow the detection of weak protein inhibitors i.e. those that only reduce DnaA activity, to normal or near normal levels (i.e. to levels comparable to wild-type DnaA activity or to activity of the DnaA protein prior to introduction of the lethal overactivity (“cos”) mutation (i.e. the “source” or “origin” or “parental” protein into which the lethal overactivity mutation is introduced)), such that there is sufficient DnaA activity for the cells to survive but the DnaA activity is not high enough to cause over-initiation, that leads to the death of the cells. It can be seen that such a screen would not allow a distinction to be made between the presence in the sample of a strong protein inhibitor which would severely reduce target protein, e.g. DnaA levels and cause cells to die due to lack of initiation of DNA replication, and the absence in the sample of any inhibitor, which causes the cells to die due to over-initiation. An analogous situation can be envisaged for other target proteins, and their lethal-overactivity mutants.
As used herein “mutation” refers to the changes in nucleotide sequence and “mutant” refers to the gene or gene product, or cell containing such a mutation.
Thus, even when a mutation is identified that enables the use of a “positive” screen i.e. a screen whereby the presence of a protein inhibitor is indicated by an increase in cell viability, rather than a decrease, it can be seen that such screens are not always suitable to identify protein inhibitors over a full range of potency. The relative rarity of lethal overactivity mutants which cause cell death by virtue of their increased activity, such as DnaAcos (or analogous over-activity mutations in other DNA replication initiator proteins), in combination with this fact, means that it was not immediately apparent, or straightforward, how to devise an effective positive screen for protein inhibitors.
To increase the efficacy of this type of screen and hence the range of inhibitors that may be identified, the present inventors have identified a mechanism whereby the use of a second mutation in the cell containing the first (i.e. lethal overactivity) mutation compensates for any severe reduction in activity of the target lethal over-activity mutation protein that may be caused by the presence of a strong protein inhibitor in a test sample. This second mutation does not affect normal cell growth or the lethal overactivity of the first mutation, however its presence compensates for any severe reduction in activity of the target protein caused by the presence of a strong protein inhibitor in the test sample. The second mutation thus salvages, or rescues, the test strain from a severe or total reduction of activity in the target protein. In this way, the inventors have been able to achieve a reliable and effective positive screen or assay for a protein inhibitor.
The rnh gene of E. coli encodes RNaseH. This enzyme functions to cleave and thereby degrade RNA in DNA:RNA hybrids. Thus, a cell containing a functional inactivation of rnh through e.g. mutation or deletion of the gene will contain persisting RNA:DNA hybrids, and the presence of such hybrids permits initiation of replication to occur independently of DnaA, and independently of the chromosomal origin OriC. Initiation of replication thus occurs from these RNA:DNA hybrids, which do not persist in cells that contain a functional RNaseH enzyme. In this way, initiation of replication does not require OriC or DnaA, and proceeds in the absence of a fully active wild-type initiation system. For example, partially functional rnh mutants are known to permit the growth of mutants that are incapable of using OriC, (Taya and Crouch, 1991, Mol. Gen. Genet., 227: 433-437; Kogoma and von Meyenburg, 1983, EMBO J. 2: 463-8).
The combination of the two mutations, DnaAcos and a deletion in rnh in E. coli (or indeed in other organisms) has not been achieved previously.
In order for cells (e.g. a population of cells) containing these two mutations to be propagated, the lethal overactivity mutant should be “inducible” i.e. capable of being “turned on” or “switched on” or expressed in particular conditions only, such that its expression may be controlled, or the phenotype is only seen when in the induced state. The cells are cultured (e.g. the cell population is expanded and maintained in culture) under conditions whereby the lethal overactivity is not induced. The assay is then performed under conditions whereby the lethal overactivity is induced. This may for example require that the mutated gene is placed under the control of an inducible promoter or the mutation may be a temperature or cold sensitive mutation, such as DnaAcos, or other conditional mutation.
Prior art methods are thus lacking that describe positive screens for protein inhibitors and antibiotics, and that allow detection of a full range of inhibitors.